Triazole macrocycle systems

ABSTRACT

The present invention provides novel peptidomimetic macrocycles and methods for their preparation and use, as well as amino acid analogs and macrocycle-forming linkers, and kits useful in their production.

CROSS-REFERENCE

This application is a continuation of U.S. Utility application Ser. No.14/677,679, filed Apr. 2, 2015, which is a continuation of U.S. Utilityapplication Ser. No. 13/370,057, filed Feb. 9, 2012, now U.S. Pat. No.9,023,988, which is a continuation of U.S. Utility application Ser. No.13/097,930, filed Apr. 29, 2011, now U.S. Pat. No. 8,637,686, which is adivisional of U.S. Utility application Ser. No. 12/037,041, filed Feb.25, 2008, now U.S. Pat. No. 7,981,999, which claims the benefit of U.S.Provisional Application 60/903,073, filed Feb. 23, 2007, each of whichis incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2017, isnamed 35224707304_2.txt and is 35,509 bytes in size.

BACKGROUND OF THE INVENTION

Peptides are becoming increasingly important in pharmaceuticalapplications. Unmodified peptides often suffer from poor metabolicstability, poor cell penetrability, and promiscuous binding due toconformational flexibility. To improve these properties, researchershave generated cyclic peptides and peptidomimetics by a variety ofmethods, including disulfide bond formation, amide bond formation, andcarbon-carbon bond formation (Jackson et al. (1991), J. Am. Chem. Soc.113:9391-9392; Phelan et al. (1997), J. Am. Chem. Soc. 119:455-460;Taylor (2002), Biopolymers 66: 49-75; Brunel et al. (2005), Chem.Commun. (20):2552-2554; Hiroshige et al. (1995), J. Am. Chem. Soc. 117:11590-11591; Blackwell et al. (1998), Angew. Chem. Int. Ed.37:3281-3284; Schafmeister et al. (2000), J. Am. Chem. Soc.122:5891-5892). Limitations of these methods include poor metabolicstability (disulfide and amide bonds), poor cell penetrability(disulfide and amide bonds), and the use of potentially toxic metals(for carbon-carbon bond formation). Thus, there is a significant needfor improved methods to produce peptides or peptidomimetics that possessincreased conformational rigidity, metabolic stability and cellpenetrability. The present invention addresses these and other needs inthe art.

SUMMARY OF THE INVENTION

Provided herein are peptidomimetic macrocycles of Formula I, wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula

L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄—]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v is an integer from 1-1000;

w is an integer from 1-1000;

x is an integer from 0-10;

y is an integer from 0-10;

z is an integer from 0-10; and

n is an integer from 1-5.

In some embodiments, L is

In other embodiments, L is

In further embodiments, at least one of R₁ and R₂ is alkyl, alkenyl,alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-. In relatedembodiments, R₁ and R₂ are independently alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-. For example,at least one of R₁ and R₂ may be alkyl, unsubstituted or substitutedwith halo-. In some embodiments, both R₁ and R₂ are independently alkyl,unsubstituted or substituted with halo-. In other embodiments, at leastone of R₁ and R₂ is methyl. In still other embodiments, both R₁ and R₂are methyl.

In some embodiments, at least one of D and E is a natural or unnaturalamino acid substituted with a high molecular weight lipid orhydrocarbon. In other embodiments, at least one of D and E is attachedto an additional macrocycle-forming linker. In still other embodiments,a secondary structure of the peptidomimetic macrocycle is more stablethan a corresponding secondary structure of a correspondingnon-macrocyclic polypeptide. In some embodiments, the peptidomimeticmacrocycle comprises an α-helix. The α-helix may comprise, for example,from 1 turn to 5 turns. In other embodiments, the α-helix is more stablethan an α-helix of a corresponding non-macrocyclic polypeptide. Themacrocycle-forming linker may span, for example, from 1 turn to 5 turnsof the α-helix, such as approximately 1, 2, 3, 4 or 5 turns of theα-helix. The length of the macrocycle-forming linker may be, forexample, about 5 Å to about 9 Å per turn of the α-helix. In someembodiments, the macrocycle-forming linker spans approximately 1 turn ofthe α-helix. In such embodiments, the length of the macrocycle-forminglinker may be approximately equal to the length of from about 6carbon-carbon bonds to about 14 carbon-carbon bonds, or may be equal tothe length of from about 8 carbon-carbon bonds to about 12 carbon-carbonbonds. In related embodiments, the the macrocycle may comprise a ring ofabout 18 atoms to 26 atoms.

In other embodiments, the macrocycle-forming linker spans approximately2 turns of the α-helix. In some embodiments, the length of themacrocycle-forming linker may be approximately equal to the length offrom about 8 carbon-carbon bonds to about 16 carbon-carbon bonds, or maybe approximately equal to the length of from about 10 carbon-carbonbonds to about 13 carbon-carbon bonds. In related embodiments, themacrocycle may comprise a ring of about 29 atoms to about 37 atoms.

In some embodiments, an amino acid useful in the synthesis of thepeptidomimetic macrocycle of the invention is a compound of Formula IIaor IIb:

wherein

R₁ and R₂ are independently alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-;

each Q and T is independently selected from the group consisting ofalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and[—R₄—K—R₄—]_(n), each of which is unsubstituted or substituted with R₅;

K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl;

R₁₀ and R₁₁ are independently —H or any protecting group suitable forpeptide synthesis;

g and h are each independently an integer from 0 to 5, wherein g+h isgreater than 1; and

n is an integer from 1 to 5.

In some embodiments, the compound is a compound of Formula IIa and R₁ isalkyl, unsubstituted or substituted with halo-. In other embodiments,the compound is a compound of Formula IIb and R₂ is alkyl, unsubstitutedor substituted with halo-. In yet other embodiments, the compound is acompound of Formula IIa and R₁ is unsubstituted alkyl. For example, R₁may be methyl. In still other embodiments, the compound is a compound ofFormula IIb and R₂ is unsubstituted alkyl. For example, R₂ may bemethyl. In some embodiments of the compounds of the invention, at leastone of R₉ and R₁₀ is a protected group suitable for peptide synthesis.In another aspect, the present invention further provides kitscomprising compounds of the invention or other amino acid analogs usefulin the preparation of the peptidomimetic macrocycles of the inventionalong with macrocyclization reagents as described herein.

In some embodiments, the invention provides a kit comprising:

a) at least one compound selected from the group consisting of compoundsof Formulas IIa and IIb:

wherein

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-;

each Q and T is independently selected from the group consisting ofalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and[—R₄—K—R₄—]_(n), each of which is unsubstituted or substituted with R₅;

K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl;

R₁₀ and R₁₁ are independently —H or any protecting group suitable forpeptide synthesis;

g and h are each independently an integer from 0 to 5;

n is an integer from 1 to 5; and

b) a macrocyclization reagent.

In some embodiments, the kit comprises a compound of Formula IIa and R₁is alkyl, unsubstituted or substituted with halo-. In relatedembodiments, R₁ is unsubstituted alkyl. For example, R₁ may be methyl.In other embodiments, the kit comprises a compound of Formula IIb and R₂is alkyl, unsubstituted or substituted with halo-. In relatedembodiments, R₂ is unsubstituted alkyl. For example, R₂ may be methyl.

In some embodiments, a kit comprises at least one compound of FormulaIIa and at least one compound of Formula IIb. A kit of the invention mayalso comprise a compound of Formula IIa or Formula IIb wherein at leastone of R₉ and R₁₀ is a protected group suitable for peptide synthesis.In specific embodiments of the kit of the invention, themacrocyclization reagent is a Cu reagent. In yet other embodiments ofthe kit of the invention, the macrocyclization reagent is a Ru reagent.

The present invention also provides a method for synthesizing apeptidomimetic macrocycle, the method comprising the steps of contactinga peptidomimetic precursor of Formula III or Formula IV:

with a macrocyclization reagent;

wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, L₁ and L₂ are asdefined above; R₁₂ is —H when the macrocyclization reagent is a Cureagent and R₁₂ is —H or alkyl when the macrocyclization reagent is a Rureagent; and further wherein said contacting step results in a covalentlinkage being formed between the alkyne and azide moiety in Formula IIIor Formula IV. For example, R₁₂ may be methyl when the macrocyclizationreagent is a Ru reagent.

In some embodiments of the method of the invention, at least one of R₁and R₂ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-. In related embodiments, R₁ and R₂ areindependently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-.

For instance, at least one of R₁ and R₂ may be alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl. The macrocyclization reagent may be a Cu reagent.Alternatively, the macrocyclization reagent may be a Ru reagent.

In some embodiments, the peptidomimetic precursor is purified prior tothe contacting step. In other embodiments, the peptidomimetic macrocycleis purified after the contacting step. In still other embodiments, thepeptidomimetic macrocycle is refolded after the contacting step. Themethod may be performed in solution, or, alternatively, the method maybe performed on a solid support.

Also envisioned herein is performing the method of the invention in thepresence of a target macromolecule that binds to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. In some embodiments, the method is performed in the presence ofa target macromolecule that binds preferentially to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. The method may also be applied to synthesize a library ofpeptidomimetic macrocycles.

The peptidomimetic macrocycle resulting from a method of the inventionmay comprise an α-helix in aqueous solution. For example, thepeptidomimetic macrocycle may exhibit increased α-helical structure inaqueous solution compared to a corresponding non-macrocyclicpolypeptide. In some embodiments, the peptidomimetic macrocycle exhibitsincreased thermal stability compared to a corresponding non-macrocyclicpolypeptide. In other embodiments, the peptidomimetic macrocycleexhibits increased biological activity compared to a correspondingnon-macrocyclic polypeptide. In still other embodiments, thepeptidomimetic macrocycle exhibits increased resistance to proteolyticdegradation compared to a corresponding non-macrocyclic polypeptide. Inyet other embodiments, the the peptidomimetic macrocycle exhibitsincreased ability to penetrate living cells compared to a correspondingnon-macrocyclic polypeptide.

In some embodiments, the alkyne moiety of the peptidomimetic precursorof Formula III or Formula IV is a sidechain of an amino acid selectedfrom the group consisting of L-propargylglycine, D-propargylglycine,(S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoicacid, (S)-2-amino-2-methyl-5-hexynoic acid,(R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoicacid, (R)-2-amino-2-methyl-6-heptynoic acid,(S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoicacid, (S)-2-amino-2-methyl-8-nonynoic acid,(R)-2-amino-2-methyl-8-nonynoic acid and the azide moiety of thepeptidomimetic precursor of Formula III or Formula IV is a sidechain ofan amino acid selected from the group consisting of ε-azido-L-lysine,ε-azido-D-lysine, ε-azido-alpha-methyl-L-lysine,ε-azido-alpha-methyl-D-lysine, δ-azido-alpha-methyl-L-ornithine, andδ-azido-alpha-methyl-D-ornithine.

In some embodiments, x+y+z is 3, and and A, B and C are independentlynatural or non-natural amino acids. In other embodiments, x+y+z is 6,and and A, B and C are independently natural or non-natural amino acids.

In some embodiments, the macrocyclization reagent is a Cu reagent andthe contacting step is performed in a solvent selected from the groupconsisting of protic solvent, aqueous solvent, organic solvent, andmixtures thereof. For example, the solvent may be chosen from the groupconsisting of H₂O, THF/H₂O, tBuOH/H₂O, DMF, DIPEA, CH₃CN, CH₂Cl₂,ClCH₂CH₂Cl or a mixture thereof. In other embodiments, themacrocyclization reagent is a Ru reagent and the contacting step isperformed in an organic solvent. For example, the solvent may be DMF,THF, CH₃CN, CH₂Cl₂ or ClCH₂CH₂Cl. The solvent may be a solvent whichfavors helix formation.

In some embodiments, the peptidomimetic macrocycle resulting fromperforming the method of the invention has the Formula (I):

wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, and L are asdefined above.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” refers to acompound comprising a plurality of amino acid residues joined by aplurality of peptide bonds and at least one macrocycle-forming linkerwhich forms a macrocycle between the α carbon of one naturally-occurringamino acid residue or non-naturally-occurring amino acid residue oramino acid analog residue and the α carbon of anothernaturally-occurring amino acid residue or non-naturally-occurring aminoacid residue or amino acid analog residue. The peptidomimeticmacrocycles optionally include one or more non-peptide bonds between oneor more amino acid residues and/or amino acid analog residues, andoptionally include one or more non-naturally-occurring amino acidresidues or amino acid analog residues in addition to any which form themacrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptidomimetic macrocycleof the invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenanceof α helical structure by a peptidomimetic macrocycle of the inventionas measured by circular dichroism. For example, in some embodiments, thepeptidomimetic macrocycles of the invention exhibit at least a 1.25,1.5, 1.75 or 2-fold increase in α-helicity as determined by circulardichroism compared to a corresponding non-macrocyclic polypeptide.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to a carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptidomimetic macrocycle. Amino acid analogsinclude, without limitation, compounds which are structurally identicalto an amino acid, as defined herein, except for the inclusion of one ormore additional methylene groups between the amino and carboxyl group(e.g., α-amino β-carboxy acids), or for the substitution of the amino orcarboxy group by a similarly reactive group (e.g., substitution of theprimary amine with a secondary or tertiary amine, or substitution or thecarboxy group with an ester).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., a BH3 domain or thep53 MDM2 binding domain) without abolishing or substantially alteringits essential biological or biochemical activity (e.g., receptor bindingor activation). An “essential” amino acid residue is a residue that,when altered from the wild-type sequence of the polypeptide, results inabolishing or substantially abolishing the polypeptide's essentialbiological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a BH3 polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family.

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,α di-substituted aminoacid).

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” as used herein refers to any reagentwhich may be used to prepare a peptidomimetic macrocycle of theinvention by mediating the reaction between an azide and alkyne. Suchreagents include, without limitation, Cu reagents such as reagents whichprovide a reactive Cu(I) species, such as CuBr, CuI or CuOTf, as well asCu(II) salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be convertedin situ to an active Cu(I) reagent by the addition of a reducing agentsuch as ascorbic acid or sodium ascorbate. Macrocyclization reagentsadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃ and—C(CH₃)₂CH₂C(O)NH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refersto an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

Peptidomimetic Macrocycles of the Invention

The present invention provides a peptidomimetic macrocycle of Formula(I):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula

L₁, L₂ and L₃ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v is an integer from 1-1000;

w is an integer from 1-1000;

x is an integer from 0-10;

y is an integer from 0-10;

z is an integer from 0-10; and

n is an integer from 1-5.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding.

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker L from a firstCα to a second Cα is selected to increase the stability of an α-helix.In some embodiments, the macrocycle-forming linker spans from 1 turn to5 turns of the α-helix. In some embodiments, the macrocycle-forminglinker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turnsof the α-helix. In some embodiments, the length of themacrocycle-forming linker is approximately 5 Å to 9 Å per turn of theα-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelength is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow:

In other embodiments, D and/or E are further modified in order tofacilitate cellular uptake. In some embodiments, lipidating orPEGylating a peptidomimetic macrocycle facilitates cellular uptake,increases bioavailability, increases blood circulation, alterspharmacokinetics, decreases immunogenicity and/or decreases the neededfrequency of administration.

In one embodiment, a peptidomimetic macrocycle exhibits improvedbiological properties such as increased structural stability, increasedaffinity for a target, increased resistance to proteolytic degradationand/or increased cell penetrability when compared to a correspondingnon-macrocyclic polypeptide. In another embodiment, a peptidomimeticmacrocycle comprises one or more α-helices in aqueous solutions and/orexhibits an increased degree of α-helicity in comparison to acorresponding non-macrocyclic polypeptide. In some embodiments, amacrocycle-forming linker increases cell permeability of thepeptidomimetic macrocycle. Without wishing to be bound by theory, it ishypothesized that the macrocycle-forming linker may increase the overallhydrophobicity of the peptidomimetic macrocycle relative to acorresponding non-macrocyclic polypeptide.

Any protein or polypeptide with a known primary amino acid sequencewhich contains a secondary structure believed to impart biologicalactivity is the subject of the present invention. For example, thesequence of the polypeptide can be analyzed and azide-containing andalkyne-containing amino acid analogs of the invention can be substitutedat the appropriate positions. The appropriate positions are determinedby ascertaining which molecular surface(s) of the secondary structure is(are) required for biological activity and, therefore, across whichother surface(s) the macrocycle forming linkers of the invention canform a macrocycle without sterically blocking the surface(s) requiredfor biological activity. Such determinations are made using methods suchas X-ray crystallography of complexes between the secondary structureand a natural binding partner to visualize residues (and surfaces)critical for activity; by sequential mutagenesis of residues in thesecondary structure to functionally identify residues (and surfaces)critical for activity; or by other methods. By such determinations, theappropriate amino acids are substituted with the amino acids analogs andmacrocycle-forming linkers of the invention. For example, for anα-helical secondary structure, one surface of the helix (e.g., amolecular surface extending longitudinally along the axis of the helixand radially 45-135° about the axis of the helix) may be required tomake contact with another biomolecule in vivo or in vitro for biologicalactivity. In such a case, a macrocycle-forming linker is designed tolink two α-carbons of the helix while extending longitudinally along thesurface of the helix in the portion of that surface not directlyrequired for activity.

In some embodiments of the invention, the peptide sequence is derivedfrom the BCL-2 family of proteins. The BCL-2 family is defined by thepresence of up to four conserved BCL-2 homology (BH) domains designatedBH1, BH2, BH3, and BH4, all of which include α-helical segments(Chittenden et al. (1995), EMBO 14:5589; Wang et al. (1996), Genes Dev.10:2859). Anti-apoptotic proteins, such as BCL-2 and BCL-X_(L), displaysequence conservation in all BH domains. Pro-apoptotic proteins aredivided into “multidomain” family members (e.g., BAK, BAX), whichpossess homology in the BH1, BH2, and BH3 domains, and “BH3-domain only”family members (e.g., BID, BAD, BIM, BIK, NOXA, PUMA), that containsequence homology exclusively in the BH3 amphipathic α-helical segment.BCL-2 family members have the capacity to form homo- and heterodimers,suggesting that competitive binding and the ratio between pro- andanti-apoptotic protein levels dictates susceptibility to death stimuli.Anti-apoptotic proteins function to protect cells from pro-apoptoticexcess, i.e., excessive programmed cell death. Additional “security”measures include regulating transcription of pro-apoptotic proteins andmaintaining them as inactive conformers, requiring either proteolyticactivation, dephosphorylation, or ligand-induced conformational changeto activate pro-death functions. In certain cell types, death signalsreceived at the plasma membrane trigger apoptosis via a mitochondrialpathway. The mitochondria can serve as a gatekeeper of cell death bysequestering cytochrome c, a critical component of a cytosolic complexwhich activates caspase 9, leading to fatal downstream proteolyticevents. Multidomain proteins such as BCL-2/BCL-X_(L) and BAK/BAX playdueling roles of guardian and executioner at the mitochondrial membrane,with their activities further regulated by upstream BH3-only members ofthe BCL-2 family. For example, BID is a member of the BH3-domain onlyfamily of pro-apoptotic proteins, and transmits death signals receivedat the plasma membrane to effector pro-apoptotic proteins at themitochondrial membrane. BID has the capability of interacting with bothpro- and anti-apoptotic proteins, and upon activation by caspase 8,triggers cytochrome c release and mitochondrial apoptosis. Deletion andmutagenesis studies determined that the amphipathic α-helical BH3segment of pro-apoptotic family members may function as a death domainand thus may represent a critical structural motif for interacting withmultidomain apoptotic proteins. Structural studies have shown that theBH3 helix can interact with anti-apoptotic proteins by inserting into ahydrophobic groove formed by the interface of BH1, 2 and 3 domains.Activated BID can be bound and sequestered by anti-apoptotic proteins(e.g., BCL-2 and BCL-X_(L)) and can trigger activation of thepro-apoptotic proteins BAX and BAK, leading to cytochrome c release anda mitochondrial apoptosis program. BAD is also a BH3-domain onlypro-apoptotic family member whose expression triggers the activation ofBAX/BAK. In contrast to BID, however, BAD displays preferential bindingto anti-apoptotic family members, BCL-2 and BCL-X_(L). Whereas the BADBH3 domain exhibits high affinity binding to BCL-2, BAD BH3 peptide isunable to activate cytochrome c release from mitochondria in vitro,suggesting that BAD is not a direct activator of BAX/BAK. Mitochondriathat over-express BCL-2 are resistant to BID-induced cytochrome crelease, but co-treatment with BAD can restore BID sensitivity.Induction of mitochondrial apoptosis by BAD appears to result fromeither: (1) displacement of BAX/BAK activators, such as BID and BID-likeproteins, from the BCL-2/BCL-XL binding pocket, or (2) selectiveoccupation of the BCL-2/BCL-XL binding pocket by BAD to preventsequestration of BID-like proteins by anti-apoptotic proteins. Thus, twoclasses of BH3-domain only proteins have emerged, BID-like proteins thatdirectly activate mitochondrial apoptosis, and BAD-like proteins, thathave the capacity to sensitize mitochondria to BID-like pro-apoptoticsby occupying the binding pockets of multidomain anti-apoptotic proteins.Various α-helical domains of BCL-2 family member proteins amendable tothe methodology disclosed herein have been disclosed (Walensky et al.(2004), Science 305:1466; and Walensky et al., U.S. Patent PublicationNo. 2005/0250680, the entire disclosures of which are incorporatedherein by reference).

In other embodiments, the peptide sequence is derived from the tumorsuppressor p53 protein which binds to the oncogene protein MDM2. TheMDM2 binding site is localized within a region of the p53 tumorsuppressor that forms an α helix. In U.S. Pat. No. 7,083,983, the entirecontents of which are incorporated herein by reference, Lane et al.disclose that the region of p53 responsible for binding to MDM2 isrepresented approximately by amino acids 13-31 (PLSQETFSDLWKLLPENNV)(SEQ ID NO: 1) of mature human P53 protein. Other modified sequencesdisclosed by Lane are also contemplated in the instant invention.Furthermore, the interaction of p53 and MDM2 has been discussed by Shairet al. (1997), Chem. & Biol. 4:791, the entire contents of which areincorporated herein by reference, and mutations in the p53 gene havebeen identified in virtually half of all reported cancer cases. Asstresses are imposed on a cell, p53 is believed to orchestrate aresponse that leads to either cell-cycle arrest and DNA repair, orprogrammed cell death. As well as mutations in the p53 gene that alterthe function of the p53 protein directly, p53 can be altered by changesin MDM2. The MDM2 protein has been shown to bind to p53 and disrupttranscriptional activation by associating with the transactivationdomain of p53. For example, an 11 amino-acid peptide derived from thetransactivation domain of p53 forms an amphipathic α-helix of 2.5 turnsthat inserts into the MDM2 crevice. Thus, in some embodiments, novelα-helix structures generated by the method of the present invention areengineered to generate structures that bind tightly to the helixacceptor and disrupt native protein-protein interactions. Thesestructures are then screened using high throughput techniques toidentify optimal small molecule peptides. The novel structures thatdisrupt the MDM2 interaction are useful for many applications,including, but not limited to, control of soft tissue sarcomas (whichover-expresses MDM2 in the presence of wild type p53). These cancers arethen, in some embodiments, held in check with small molecules thatintercept MDM2, thereby preventing suppression of p53. Additionally, insome embodiments, small molecules disrupters of MDM2-p53 interactionsare used as adjuvant therapy to help control and modulate the extent ofthe p53 dependent apoptosis response in conventional chemotherapy.

A non-limiting exemplary list of suitable peptide sequences for use inthe present invention is given below:

TABLE 1 Sequence Cross-linked Sequence Name (bold = critical residues) (X  = x-link residue) BH3 peptides (SEQ ID NOS 2-24) (SEQ ID NOS 25-27)BID-BH3 QEDIIRNIARHLAQVGDSMDRSIPP QEDIIRNIARHLA X VGD X MDRSIPP BIM-BH3DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIAQELR X IGD X FNAYYAR BAD-BH3NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRYGRELR X MSD X FVDSFKK PUMA-BH3EEQWAREIGAQLRRMADDLNAQYER EEQWAREIGAQLR X MAD X LNAQYER Hrk-BH3RSSAAQLTAARLKALGDELHQRTM RSSAAQLTAARLK X LGD X LHQRTM NOXAA-BH3AELPPEFAAQLRKIGDKVYCTW AELPPEFAAQLR X IGD X VYCTW NOXAB-BH3VPADLKDECAQLRRIGDKVNLRQKL VPADLKDECAQLR X IGD X VNLRQKL BMF-BH3QHRAEVQIARKLQCIADQFHRLHT QHRAEVQIARKLQ X IAD X FHRLHT BLK-BH3SSAAQLTAARLKALGDELHQRT SSAAQLTAARLK X LGD X LHQRT BIK-BH3CMEGSDALALRLACIGDEMDVSLRA CMEGSDALALRLA X IGD X MDVSLRA Bnip3DIERRKEVESILKKNSDWIWDWSS DIERRKEVESILK X NSD X IWDWSS BOK-BH3GRLAEVCAVLLRLGDELEMIRP GRLAEVCAVLL X LGD X LEMIRP BAX-BH3PQDASTKKSECLKRIGDELDSNMEL PQDASTKKSECLK X IGD X LDSNMEL BAK-BH3PSSTMGQVGRQLAIIGDDINRR PSSTMGQVGRQLA X IGD X INRR BCL2L1-BH3KQALREAGDEFELR KQALR X AGD X FELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRRLSPPVVHLALALR X AGD X FSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRYEVIPMAAVKQALR X AGD X FELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPLHQAMR XAGD X FETRF MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA ATSRKLETLR X VGD X VQRNHETAMTD-BH3 LAEVCTVLLRLGDELEQIR LAEVCTVLL X LGD X LEQIR MAP-1-BH3MTVGELSRALGHENGSLDP MTVGELSRALG X ENG X LDP NIX-BH3VVEGEKEVEALKKSADWVSDWS VVEGEKEVEALK X SAD X VSDWS 4ICD(ERBB4)-BH3SMARDPQRYLVIQGDDRMKL SMARDPQRYLV X QGD X RMKL

-   Table 1 lists human sequences which target the BH3 binding site and    are implicated in cancers, autoimmune disorders, metabolic diseases    and other human disease conditions.

TABLE 2 Sequence Cross-linked Sequence Name (bold = critical residues) (X  = x-link residue) BH3 peptides (SEQ ID NOS 2-24) (SEQ ID NOS 48-70)BID-BH3 QEDIIRNIARHLAQVGDSMDRSIPP QEDIIRNI X RHL X VGD S MDRSIPP BIM-BH3DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIAQELR X IGD X FNAYYAR BAD-BH3NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRY X REL X RMSDFVDSFKK PUMA-BH3EEQWAREIGAQLRRMADDLNAQYER EEQWAREI X AQL X RMADDLNAQYER Hrk-BH3RSSAAQLTAARLKALGDELHQRTM RSSAAQLT X ARL X ALGDELHQRTM NOXAA-BH3AELPPEFAAQLRKIGDKVYCTW AELPPEF X AQLR X KIGDKVYCTW NOXAB-BH3VPADLKDECAQLRRIGDKVNLRQKL VPADLKDE X AQL X RIGDKVNLRQKL BMF-BH3QHRAEVQIARKLQCIADQFHRLHT QHRAEVQI X RKLQ X CIADFHRLHT BLK-BH3SSAAQLTAARLKALGDELHQRT SSAAQLT X ARL X ALGDELHQRT BIK-BH3CMEGSDALALRLACIGDEMDVSLRA CMEGSDAL X LRL X CIGDEMDVSLRA Bnip3DIERRKEVESILKKNSDWIWDWSS DIERRKEV X SIL X KNSDIWDWSS BOK-BH3GRLAEVCAVLLRLGDELEMIRP GRLAEV X AVLL X RLGDELEMIRP BAX-BH3PQDASTKKSECLKRIGDELDSNMEL PQDASTKK X ECL X RIGDELDSNMEL BAK-BH3PSSTMGQVGRQLAIIGDDINRR PSSTMGQV X RQL X IIGDDINRR BCL2L1-BH3KQALREAGDEFELR X QAL X EAGDEFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRRLSPPVVHL X LAL X QAGDDFSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY EVIPMAAV XQAL X EAGDEFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF PADPL X QAM X AAGDEFETRFMCL1-BH3 ATSRKLETLRRVGDGVQRNHETA ATSRK X ETL X RVGDVQRNHETA MTD-BH3LAEVCTVLLRLGDELEQIR LAEV X TVL X RLGDLEQIR MAP- 1 -BH3MTVGELSRALGHENGSLDP MTVGEL X RAL X HENGSLDP NIX-BH3VVEGEKEVEALKKSADWVSDWS VVEGEKE X EAL X KSADWVSDWS 4ICD(ERBB4)-BH3SMARDPQRYLVIQGDDRMKL SMARDP X RYL X IQGDRMKL

Table 2 lists human sequences which target the BH3 binding site and areimplicated in cancers, autoimmune disorders, metabolic diseases andother human disease conditions.

TABLE 3 Cross-linked Sequence Name Sequence ( X  = x-link residue)P53 peptides (bold = critical residues) (SEQ ID NOS 76-86)hp53 peptide_veryshort LSQETFSDLWKLLPEN X SQE X FSDLWKLLPEN(SEQ ID NO: 71) hp53 peptide_short PPLSQETFSDLWKLLPENN PP X SQE XFSDLWKLLPENN (SEQ ID NO: 72) hp53-P27S peptide-short PPLSQETFSDLWKLLSENNPP X SQE X FSDLWKLLSENN (SEQ ID NO: 73) hp53 peptide_LongDPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPP X SQE X FSDLWKLLPENNVLSPLP(SEQ ID NO: 74) hp53-P27S peptide_Long DPSVEPPLSQETFSDLWKLLSENNVLSPLPDPSVEPP X SQE X FSDLWKLLSENNVLSPLP (SEQ ID NO: 75)hp53 peptide_veryshort LSQETFSDLWKLLPEN LSQETFSDLW X LLPXN(SEQ ID NO: 71) hp53 peptide_short PPLSQETFSDLWKLLPENN PPLSQETFSDLW XLLP X NN (SEQ ID NOS: 72) hp53-P27S peptide-short PPLSQETFSDLWKLLSENNPPLSQETFSDLW X LLS X NN (SEQ ID NO: 73) hp53 peptide_LongDPSVEPPLSQETFSDLWKLLPENNVLSPLP DPSVEPPLSQETFSDLW X LLP X NNVLSPLP(SEQ ID NO: 74) hp53-P27S peptide_Long DPSVEPPLSQETFSDLWKLLSENNVLSPLPDPSVEPPLSQETFSDLW X LLS X NNVLSPLP (SEQ ID NO: 75)

Table 3 lists human sequences which target the p53 binding site ofMDM2/X and are implicated in cancers.

TABLE 4 Sequence Cross-linked Sequence Name (bold = critical residues) (X  = x-link residue) GPCR peptide ligands (SEQ ID NOS 86-91)(SEQ ID NOS 92-97) Angiotensin II DRVYIHPF DR X Y X HPF BombesinEQRLGNQWAVGHLM EQRLGN X WAVGHL X Bradykinin RPPGFSPFR RPP X FSPFR X C5aISHKDMQLGR ISHKDM X LGR X C3a ARASHLGLAR ARASHL X LAR X α-melanocyte SYSMEHFRWGKPV SYSM X HFRW X KPV stimulating hormone

Table 4 lists sequences which target human G protein-coupled receptorsand are implicated in numerous human disease conditions (Tyndall et al.(2005), Chem. Rev. 105:793-826).

Methods of Preparing the Peptidomimetic Macrocycles of the Invention

Methods of synthesizing the peptidomimetic macrocycles of the inventionare disclosed herein. In some embodiments, the synthesis of thesepeptidomimetic macrocycles involves a multi-step process that featuresthe synthesis of a peptidomimetic precursor containing an azide moietyand an alkyne moiety; followed by contacting the peptidomimeticprecursor with a macrocyclization reagent to generate a triazole-linkedpeptidomimetic macrocycle. Macrocycles or macrocycle precursors aresynthesized, for example, by solution phase or solid-phase methods, andcan contain both naturally-occurring and non-naturally-occurring aminoacids. See, for example, Hunt, “The Non-Protein Amino Acids” inChemistry and Biochemistry of the Amino Acids, edited by G. C. Barrett,Chapman and Hall, 1985.

In some embodiments, an azide is linked to the α-carbon of a residue andan alkyne is attached to the α-carbon of another residue. In someembodiments, the azide moieties are azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, alpha-methyl-D-lysine,L-ornithine, D-ornithine, alpha-methyl-L-ornithine oralpha-methyl-D-ornithine. In another embodiment, the alkyne moiety isL-propargylglycine. In yet other embodiments, the alkyne moiety is anamino acid selected from the group consisting of L-propargylglycine,D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,(R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoicacid, (R)-2-amino-2-methyl-5-hexynoic acid,(S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoicacid, (S)-2-amino-2-methyl-7-octynoic acid,(R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoicacid and (R)-2-amino-2-methyl-8-nonynoic acid.

In some embodiments, the invention provides a method for synthesizing apeptidomimetic macrocycle, the method comprising the steps of contactinga peptidomimetic precursor of Formula III or Formula IV:

with a macrocyclization reagent;

wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, L₁ and L₂ are asdefined above; R₁₂ is —H when the macrocyclization reagent is a Cureagent and R₁₂ is —H or alkyl when the macrocyclization reagent is a Rureagent; and further wherein said contacting step results in a covalentlinkage being formed between the alkyne and azide moiety in Formula IIIor Formula IV. For example, R₁₂ may be methyl when the macrocyclizationreagent is a Ru reagent.

In some embodiments of the method of the invention, at least one of R₁and R₂ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-. In related embodiments, R₁ and R₂ areindependently alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-.

For instance, at least one of R₁ and R₂ may be alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl. The macrocyclization reagent may be a Cu reagentor a Ru reagent.

In some embodiments, the peptidomimetic precursor is purified prior tothe contacting step. In other embodiments, the peptidomimetic macrocycleis purified after the contacting step. In still other embodiments, thepeptidomimetic macrocycle is refolded after the contacting step. Themethod may be performed in solution, or, alternatively, the method maybe performed on a solid support.

Also envisioned herein is performing the method of the invention in thepresence of a target macromolecule that binds to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. In some embodiments, the method is performed in the presence ofa target macromolecule that binds preferentially to the peptidomimeticprecursor or peptidomimetic macrocycle under conditions that favor saidbinding. The method may also be applied to synthesize a library ofpeptidomimetic macrocycles.

The peptidomimetic macrocycle resulting from a method of the inventionmay comprise an α-helix in aqueous solution. For example, thepeptidomimetic macrocycle may exhibit increased α-helical structure inaqueous solution compared to a corresponding non-macrocyclicpolypeptide. In some embodiments, the peptidomimetic macrocycle exhibitsincreased thermal stability compared to a corresponding non-macrocyclicpolypeptide. In other embodiments, the peptidomimetic macrocycleexhibits increased biological activity compared to a correspondingnon-macrocyclic polypeptide. In still other embodiments, thepeptidomimetic macrocycle exhibits increased resistance to proteolyticdegradation compared to a corresponding non-macrocyclic polypeptide. Inyet other embodiments, the the peptidomimetic macrocycle exhibitsincreased ability to penetrate living cells compared to a correspondingnon-macrocyclic polypeptide.

In some embodiments, the alkyne moiety of the peptidomimetic precursorof Formula III or Formula IV is a sidechain of an amino acid selectedfrom the group consisting of L-propargylglycine, D-propargylglycine,(S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoicacid, (S)-2-amino-2-methyl-5-hexynoic acid,(R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoicacid, (R)-2-amino-2-methyl-6-heptynoic acid,(S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoicacid, (S)-2-amino-2-methyl-8-nonynoic acid, and(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the azidemoiety of the peptidomimetic precursor of Formula III or Formula IV is asidechain of an amino acid selected from the group consisting ofε-azido-L-lysine, ε-azido-D-lysine, ε-azido-α-methyl-L-lysine,ε-azido-α-methyl-D-lysine, δ-azido-α-methyl-L-ornithine, andδ-azido-α-methyl-D-ornithine.

In some embodiments, x+y+z is 3, and and A, B and C are independentlynatural or non-natural amino acids. In other embodiments, x+y+z is 6,and and A, B and C are independently natural or non-natural amino acids.

In some embodiments, the contacting step is performed in a solventselected from the group consisting of protic solvent, aqueous solvent,organic solvent, and mixtures thereof. For example, the solvent may bechosen from the group consisting of H₂O, THF, THF/H₂O, tBuOH/H₂O, DMF,DIPEA, CH₃CN or CH₂Cl₂, ClCH₂CH₂Cl or a mixture thereof. The solvent maybe a solvent which favors helix formation.

In some embodiments, the peptidomimetic macrocycle resulting fromperforming the method of the invention has the Formula (I):

wherein v, w, x, y, z, A, B, C, D, E, R₁, R₂, R₇, R₈, and L are asdefined above.

Alternative but equivalent protecting groups, leaving groups or reagentsare substituted, and certain of the synthetic steps are performed inalternative sequences or orders to produce the desired compounds.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein include, for example, those such as described inLarock, Comprehensive Organic Transformations, VCH Publishers (1989);Greene and Wuts, Protective Groups in Organic Synthesis, 2d. Ed., JohnWiley and Sons (1991); Fieser and Fieser, Fieser and Fieser's Reagentsfor Organic Synthesis, John Wiley and Sons (1994); and Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The peptidomimetic macrocycles of the invention are made, for example,by chemical synthesis methods, such as described in Fields et al.,Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H.Freeman & Co., New York, N.Y., 1992, p. 77. Hence, for example, peptidesare synthesized using the automated Merrifield techniques of solid phasesynthesis with the amine protected by either tBoc or Fmoc chemistryusing side chain protected amino acids on, for example, an automatedpeptide synthesizer (e.g., Applied Biosystems (Foster City, Calif.),Model 430A, 431, or 433).

One manner of producing the peptidomimetic precursors and peptidomimeticmacrocycles described herein uses solid phase peptide synthesis (SPPS).The C-terminal amino acid is attached to a cross-linked polystyreneresin via an acid labile bond with a linker molecule. This resin isinsoluble in the solvents used for synthesis, making it relativelysimple and fast to wash away excess reagents and by-products. TheN-terminus is protected with the Fmoc group, which is stable in acid,but removable by base. Side chain functional groups are protected asnecessary with base stable, acid labile groups.

Longer peptidomimetic precursors are produced, for example, byconjoining individual synthetic peptides using native chemical ligation.Alternatively, the longer synthetic peptides are biosynthesized by wellknown recombinant DNA and protein expression techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptidomimetic precursor of this invention,the amino acid sequence is reverse translated to obtain a nucleic acidsequence encoding the amino acid sequence, preferably with codons thatare optimum for the organism in which the gene is to be expressed. Next,a synthetic gene is made, typically by synthesizing oligonucleotideswhich encode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods.

The peptidomimetic precursors are made, for example, in ahigh-throughput, combinatorial fashion using, for example, ahigh-throughput polychannel combinatorial synthesizer (e.g., Model Apex396 multichannel peptide synthesizer from AAPPTEC, Inc., Louisville,Ky.).

The following synthetic schemes are provided solely to illustrate thepresent invention and are not intended to limit the scope of theinvention, as described herein. To simplify the drawings, theillustrative schemes depict azido amino acid analogsε-azido-α-methyl-L-lysine and ε-azido-α-methyl-D-lysine, and alkyneamino acid analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoicacid, and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the followingsynthetic schemes, each R₁, R₂, R₇ and R₈ is —H; each L₁ is —(CH₂)₄—;and each L₂ is —(CH₂)—. However, as noted throughout the detaileddescription above, many other amino acid analogs can be employed inwhich R₁, R₂, R₇, R₈, L₁ and L₂ can be independently selected from thevarious structures disclosed herein.

Synthetic Scheme 1 describes the preparation of several compounds of theinvention. Ni(II) complexes of Schiff bases derived from the chiralauxiliary (S)-2-[N—(N′-benzylprolyl)amino]benzophenone (BPB) and aminoacids such as glycine or alanine are prepared as described in Belokon etal. (1998), Tetrahedron Asymm. 9:4249-4252. The resulting complexes aresubsequently reacted with alkylating reagents comprising an azido oralkynyl moiety to yield enantiomerically enriched compounds of theinvention. If desired, the resulting compounds can be protected for usein peptide synthesis.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 2, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aCu(I) in organic or aqueous solutions (Rostovtsev et al. (2002), Angew.Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem.67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783;Purina et al. (2005), Angew. Chem. Int. Ed. 44:2215-2220). In oneembodiment, the triazole forming reaction is performed under conditionsthat favor α-helix formation. In one embodiment, the macrocyclizationstep is performed in a solvent chosen from the group consisting of H₂O,THF, CH₃CN, DMF, DIPEA, tBuOH or a mixture thereof. In anotherembodiment, the macrocyclization step is performed in DMF. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 3, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Cu(I) reagent on the resin as a crude mixture (Rostovtsev et al.(2002), Angew. Chem. Int. Ed. 41:2596-2599; Tornoe et al. (2002), J.Org. Chem. 67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.44:2215-2220). The resultant triazole-containing peptidomimeticmacrocycle is then deprotected and cleaved from the solid-phase resin bystandard conditions (e.g., strong acid such as 95% TFA). In someembodiments, the macrocyclization step is performed in a solvent chosenfrom the group consisting of CH₂Cl₂, ClCH₂CH₂Cl, DMF, THF, NMP, DIPEA,2,6-lutidine, pyridine, DMSO, H₂O or a mixture thereof. In someembodiments, the macrocyclization step is performed in a bufferedaqueous or partially aqueous solvent.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 4, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solution-phaseor solid-phase peptide synthesis (SPPS) using the commercially availableamino acid N-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected formsof the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is then deprotected and cleaved from thesolid-phase resin by standard conditions (e.g., strong acid such as 95%TFA). The peptidomimetic precursor is reacted as a crude mixture or ispurified prior to reaction with a macrocyclization reagent such as aRu(II) reagents, for example Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen etal. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem.Soc. 127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of DMF, CH₃CNand THF.

In the general method for the synthesis of peptidomimetic macrocyclesshown in Synthetic Scheme 5, the peptidomimetic precursor contains anazide moiety and an alkyne moiety and is synthesized by solid-phasepeptide synthesis (SPPS) using the commercially available amino acidN-α-Fmoc-L-propargylglycine and the N-α-Fmoc-protected forms of theamino acids (S)-2-amino-2-methyl-4-pentynoic acid,(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine. Thepeptidomimetic precursor is reacted with a macrocyclization reagent suchas a Ru(II) reagent on the resin as a crude mixture. For example, thereagent can be Cp*RuCl(PPh₃)₂ or [Cp*RuCl]₄ (Rasmussen et al. (2007),Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am. Chem. Soc.127:15998-15999). In some embodiments, the macrocyclization step isperformed in a solvent chosen from the group consisting of CH₂Cl₂,ClCH₂CH₂Cl, CH₃CN, DMF, and THF.

Several exemplary peptidomimetic macrocycles are shown in Table 5. Forthese macrocycles, a corresponding non-macrocyclic polypeptide is theBID BH3 polypeptide sequence fragment DIIRNIARHLAQVGDSMDRSI (SEQ ID NO:98). “Nle” represents norleucine and replaces a methionine residue. Itis envisioned that similar linkers are used to synthesize peptidomimeticmacrocycles based on the polypeptide sequences disclosed in Table 1through Table 4.

Table 5

TABLE 5

MW = 2464

MW = 2464

MW = 2478

MW = 2478

MW = 2492

MW = 2492

MW = 2464

MW = 2464

MW = 2478

MW = 2478

MW = 2492

MW = 2492

Table 5 shows exemplary peptidomimetic macrocycles of the invention.“Nle” represents norleucine (peptide disclosed is SEQ ID NO: 99).

Amino Acid Analogs

The present invention contemplates the use of non-naturally-occurringamino acids and amino acid analogs in the synthesis of thepeptidomimetic macrocycles described herein. Any amino acid or aminoacid analog amenable to the synthetic methods employed for the synthesisof stable triazole containing peptidomimetic macrocycles can be used inthe present invention. For example, L-propargylglycine is contemplatedas a useful amino acid in the present invention. However, otheralkyne-containing amino acids that contain a different amino acid sidechain are also useful in the invention. For example, L-propargylglycinecontains one methylene unit between the α-carbon of the amino acid andthe alkyne of the amino acid side chain. The invention also contemplatesthe use of amino acids with multiple methylene units between theα-carbon and the alkyne. Also, the azido-analogs of amino acidsL-lysine, D-lysine, alpha-methyl-L-lysine, and alpha-methyl-D-lysine arecontemplated as useful amino acids in the present invention. However,other terminal azide amino acids that contain a different amino acidside chain are also useful in the invention. For example, theazido-analog of L-lysine contains four methylene units between theα-carbon of the amino acid and the terminal azide of the amino acid sidechain. The invention also contemplates the use of amino acids with fewerthan or greater than four methylene units between the α-carbon and theterminal azide. Table 6 shows some amino acids useful in the preparationof peptidomimetic macrocycles of the invention.

TABLE 6

N-α-Fmoc-L-propargyl glycine

N-α-Fmoc-(S)-2-amino-2- methyl-4-pentynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-5-hexynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-6-heptynoic acid

N-α-Fmoc-(S)-2-amino-2- methyl-7-octynoic acid

N-α-Fmoc-(S)-2-amino-2-methyl- 8-nonynoic acid

N-α-Fmoc-D-propargyl glycine

N-α-Fmoc-(R)-2-amino-2- methyl-4-pentynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-5-hexynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-6-heptynoic acid

N-α-Fmoc-(R)-2-amino-2- methyl-7-octynoic acid

N-α-Fmoc-(R)-2-amino-2-methyl- 8-nonynoic acid

N-α-Fmoc-ϵ-azido-L- lysine

N-α-Fmoc-ϵ-azido-α- methyl-L-lysine

N-α-Fmoc-δ-azido-L- ornithine

N-α-Fmoc-ϵ-azido-α- methyl-L-ornithine

N-α-Fmoc-ϵ-azido-D- lysine

N-α-Fmoc-ϵ-azido-α- methyl-D-lysine

N-α-Fmoc-δ-azido-D- ornithine

N-α-Fmoc-ϵ-azido-α- methyl-D-ornithine

-   -   Table 6 shows exemplary amino acids useful in the preparation of        peptidomimetic macrocycles of the invention.

In some embodiments the amino acids and amino acid analogs are of theD-configuration. In other embodiments they are of the L-configuration.In some embodiments, some of the amino acids and amino acid analogscontained in the peptidomimetic are of the D-configuration while some ofthe amino acids and amino acid analogs are of the L-configuration. Insome embodiments the amino acid analogs are α,α-disubstituted, such asα-methyl-L-propargylglycine, α-methyl-D-propargylglycine,ε-azido-alpha-methyl-L-lysine, and ε-azido-alpha-methyl-D-lysine. Insome embodiments the amino acid analogs are N-alkylated, e.g.,N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,N-methyl-ε-azido-L-lysine, and N-methyl-ε-azido-D-lysine.

In some embodiments, the —NH moiety of the amino acid is protected usinga protecting group, including without limitation -Fmoc and -Boc. Inother embodiments, the amino acid is not protected prior to synthesis ofthe peptidomimetic macrocycle.

In some embodiments, an amino acid useful in the synthesis of thepeptidomimetic macrocycle of the invention is a compound of Formula IIaor IIb:

wherein

R₁ and R₂ are independently alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-;

each Q and T is independently selected from the group consisting ofalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and[—R₄—K—R₄—]_(n), each of which is unsubstituted or substituted with R₅;

K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl;

R₁₀ and R₁₁ are independently —H or any protecting group suitable forpeptide synthesis;

g and h are each independently an integer from 0 to 5, wherein g+h isgreater than 1; and

n is an integer from 1 to 5.

In some embodiments, the compound is a compound of Formula IIa and R₁ isalkyl, unsubstituted or substituted with halo-. In other embodiments,the compound is a compound of Formula IIb and R₂ is alkyl, unsubstitutedor substituted with halo-. In yet other embodiments, the compound is acompound of Formula IIa and R₁ is unsubstituted alkyl. For example, R₁may be methyl. In still other embodiments, the compound is a compound ofFormula IIb and R₂ is unsubstituted alkyl. For example, R₂ may bemethyl.

In some embodiments of the compounds of the invention, at least one ofR₉ and R₁₀ is a protected group suitable for peptide synthesis.

Kits

In another aspect, the present invention further provides kitscomprising compounds of the invention or other amino acid analogs usefulin the preparation of the peptidomimetic macrocycles of the inventionalong with macrocyclization reagents as described herein.

In some embodiments, the invention provides a kit comprising:

a) at least one compound selected from the group consisting of compoundsof Formulas IIa and IIb:

wherein

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted orsubstituted with halo-;

each Q and T is independently selected from the group consisting ofalkylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene and[—R₄—K—R₄—]_(n), each of which is unsubstituted or substituted with R₅;

K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

R₄ is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ and R₈ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroalkylalkyl, or heterocycloalkyl;

R₁₀ and R₁₁ are independently —H or any protecting group suitable forpeptide synthesis;

g and h are each independently an integer from 0 to 5;

n is an integer from 1 to 5; and

b) a macrocyclization reagent.

In some embodiments, the kit comprises a compound of Formula IIa and R₁is alkyl, unsubstituted or substituted with halo-. In relatedembodiments, R₁ is unsubstituted alkyl. For example, R₁ may be methyl.In other embodiments, the kit comprises a compound of Formula IIb and R₂is alkyl, unsubstituted or substituted with halo-. In relatedembodiments, R₂ is unsubstituted alkyl. For example, R₂ may be methyl.

In some embodiments, a kit comprises at least one compound of FormulaIIa and at least one compound of Formula IIb. A kit of the invention mayalso comprise a compound of Formula IIa or Formula IIb wherein at leastone of R₉ and R₁₀ is a protected group suitable for peptide synthesis.In specific embodiments of the kit of the invention, themacrocyclization reagent is a Cu reagent or a Ru reagent. In someembodiments, the kit contains a plurality of compounds of Formula IIaand/or Formula IIb. In some embodiments, the kit comprises one or morecontainers holding one or more amino acid analogs as described herein.In other embodiments, the kit comprises one or more containers holdingone or more macrocyclization reagents as described herein. In yet otherembodiments, the kit comprises one or more containers holding one ormore amino acid analogs as described herein, as well as one or morecontainers holding one or more macrocyclization reagents as describedherein.

For example, in some embodiments, the kit comprises a container holdingat least two amino acid analogs, as described above, at least one havinga side-chain alkyne and at least one having a side-chain terminal azidemoiety, the amino acid analog optionally protected and suitable for thesyntheses described herein. In some embodiments, the amino acid analogis selected from the group consisting of L-propargylglycine,D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,(R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoicacid, (R)-2-amino-2-methyl-5-hexynoic acid,(S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoicacid, (S)-2-amino-2-methyl-7-octynoic acid,(R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoicacid, (R)-2-amino-2-methyl-8-nonynoic acid, ε-azido-L-lysine,ε-azido-D-lysine, ε-azido-α-methyl-L-lysine, andε-azido-α-methyl-D-lysine, δ-azido-α-methyl-L-ornithine, andδ-azido-α-methyl-D-ornithine and all forms suitably protected for liquidor solid phase peptide synthesis.

In some embodiments, the kit comprises a container holding at least onenon-naturally-occurring amino acid, or amino acid analog, bound to asolid support compatible with the syntheses described herein forpeptidomimetic macrocycles. In some embodiments, the kit comprises onecontainer holding an amino acid analog of the invention including aterminal alkyne moiety in combination with a container holding an aminoacid analog of the invention including a terminal azide moiety incombination with a macrocyclization reagent of the invention.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below. In someembodiments, a macrocycle of the invention has enhanced propertiesrelative to a corresponding non-macrocyclic polypeptide. A correspondingnon-macrocyclic polypeptide is, for example, a precursor of apeptidomimetic macrocycle, such as a compound of Formula III or IV whichis converted into said macrocycle. Alternatively, a correspondingnon-macrocyclic polypeptide is a polypeptide sequence, such as a naturalpolypeptide sequence which has substantial sequence overlap with themacrocycle of the invention. Numerous examples of natural polypeptidescorresponding to the macrocyclic polypeptide are shown in Tables 1, 2, 3and 4.

In general, a corresponding non-macrocyclic polypeptide can also be alabeled natural polypeptide or peptidomimetic precursor. Such labeling,for example by fluorescent or radioactive labeling, is used if necessaryin some of the assays described below. In such assays, both themacrocycle and the corresponding non-macrocyclic polypeptide aretypically labeled by similar or functionally equivalent methods.

Assay to Determine α-Helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, unmodified pro-apoptotic BH3 domains are predominantlyrandom coils in solution, with α-helical content usually under 25%.Peptidomimetic macrocycles with optimized linkers, on the other hand,possess, for example, an alpha-helicity that is at least two-foldgreater than that of a corresponding non-macrocyclic polypeptide. Insome embodiments, macrocycles of the invention will possess analpha-helicity of greater than 50%. To assay the helicity ofpeptidomimetic macrocyles of the invention, such as BH3 domain-basedmacrocycles, the compounds are dissolved in an aqueous solution (e.g. 50mM potassium phosphate solution at pH 7, or distilled H₂O, toconcentrations of 25-50 μM). Circular dichroism (CD) spectra areobtained on a spectropolarimeter (e.g., Jasco J-710) using standardmeasurement parameters (e.g. temperature, 20° C.; wavelength, 190-260nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helicalcontent of each peptide is calculated by dividing the mean residueellipticity (e.g. [Φ]222 obs) by the reported value for a model helicaldecapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding non-macrocyclic polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on meltine temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding non-macrocyclicpolypeptide. For example, the peptidomimetic macrocycle and acorresponding non-macrocyclic polypeptide are incubated with trypsinagarose and the reactions quenched at various time points bycentrifugation and subsequent HPLC injection to quantitate the residualsubstrate by ultraviolet absorption at 280 nm. Briefly, thepeptidomimetic macrocycle and peptidomimetic precursor (5 mcg) areincubated with trypsin agarose (Pierce) (S/E ˜125) for 0, 10, 20, 90,and 180 minutes. Reactions are quenched by tabletop centrifugation athigh speed; remaining substrate in the isolated supernatant isquantified by HPLC-based peak detection at 280 nm. The proteolyticreaction displays first order kinetics and the rate constant, k, isdetermined from a plot of ln [S] versus time (k=−1× slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding non-macrocyclic polypeptide peptide, and possess an exvivo half-life of 12 hours or more. For ex vivo serum stability studies,a variety of assays may be used. For example, a peptidomimeticmacrocycle and a corresponding non-macrocyclic polypeptide (in aspecific example, the corresponding natural polypeptide) (2 mcg) areincubated with fresh mouse, rat and/or human serum (2 mL) at 37° C. for0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound,the following procedure may be used: The samples are extracted bytransferring 100 μl of sera to 2 ml centrifuge tubes followed by theaddition of 10 μL of 50% formic acid and 500 μL acetonitrile andcentrifugation at 14,000 RPM for 10 min at 4±2° C. The supernatants arethen transferred to fresh 2 ml tubes and evaporated on Turbovap underN₂<10 psi, 37° C. The samples are reconstituted in 100 μL of 50:50acetonitrile:water and submitted to LC-MS/MS analysis.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) is used, for example. The FPA techniquemeasures the molecular orientation and mobility using polarized lightand fluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding non-macrocyclic polypeptide.

Acceptor proteins for BH3-peptides such as BCL-2, BCL-X_(L), BAX or MCL1may, for example, be used in this assay. Acceptor proteins for p53peptides such as MDM2 or MDMX may also be used in this assay.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide (e.g. a BH3 peptide or a p53 peptide) andan acceptor protein, a fluorescence polarization assay (FPA) utilizing afluoresceinated peptidomimetic macrocycle derived from a peptidomimeticprecursor sequence is used, for example. The FPA technique measures themolecular orientation and mobility using polarized light and fluorescenttracer. When excited with polarized light, fluorescent tracers (e.g.,FITC) attached to molecules with high apparent molecular weights (e.g.FITC-labeled peptides bound to a large protein) emit higher levels ofpolarized fluorescence due to their slower rates of rotation as comparedto fluorescent tracers attached to smaller molecules (e.g. FITC-labeledpeptides that are free in solution). A compound that antagonizes theinteraction between the fluoresceinated peptidomimetic macrocycle and anacceptor protein will be detected in a competitive binding FPAexperiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay. Acceptor proteins for BH3-peptides such as BCL2, BCL-XL, BAXor MCL1 can be used in this assay. Acceptor proteins for p53 peptidessuch as MDM2 or MDMX can be used in this assay.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle, includingBCL2, MCL1, BCL-XL, A1, BAX, BAK, MDM2 or MDMX.

Cellular Permeability Assays.

A peptidomimetic macrocycle is, for example, more cell permeablecompared to a corresponding non-macrocyclic polypeptide. In someembodiments, the peptidomimetic macrocycles are more cell permeable thana corresponding non-macrocyclic polypeptides. Peptidomimetic macrocycleswith optimized linkers possess, for example, cell permeability that isat least two-fold greater than a corresponding non-macrocyclicpolypeptide, and often 20% or more of the applied peptide will beobserved to have penetrated the cell after 4 hours. To measure the cellpermeability of peptidomimetic macrocycles and correspondingnon-macrocyclic polypeptides, intact cells are incubated withfluoresceinated peptidomimetic macrocycles or correspondingnon-macrocyclic polypeptides (10 μM) for 4 hrs in serum free media at37° C., washed twice with media and incubated with trypsin (0.25%) for10 min at 37° C. The cells are washed again and resuspended in PBS.Cellular fluorescence is analyzed, for example, by using either aFACSCalibur flow cytometer or Cellomics' KineticScan® HCS Reader.

Cellular Efficacy Assays.

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of peptidomimetic macrocyclesof the invention in vivo, the compounds are, for example, given alone(IP, IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4; 11 cells(established from the bone marrow of a patient with acute lymphoblasticleukemia) that stably express luciferase are injected by tail vein inNOD-SCID mice 3 hrs after they have been subjected to total bodyirradiation. If left untreated, this form of leukemia is fatal in 3weeks in this model. The leukemia is readily monitored, for example, byinjecting the mice with D-luciferin (60 mg/kg) and imaging theanesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper LifeSciences, Hopkinton, Mass.). Total body bioluminescence is quantified byintegration of photonic flux (photons/sec) by Living Image Software(Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocyclesalone or in combination with sub-optimal doses of relevantchemotherapeutics agents are, for example, administered to leukemic mice(10 days after injection/day 1 of experiment, in bioluminescence rangeof 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughoutthe experiment every other day and survival monitored daily for theduration of the experiment. Expired mice are optionally subjected tonecropsy at the end of the experiment. Another animal model isimplantation into NOD-SCID mice of DoHH2, a cell line derived from humanfollicular lymphoma, that stably expresses luciferase. These in vivotests optionally generate preliminary pharmacokinetic, pharmacodynamicand toxicology data.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycleof the invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of thepeptidomimetic macrocycles of the invention can thus be evaluated byperforming comparisons of the patient groups with respect to factorssuch as survival and quality-of-life. In this example, the patient grouptreated with a peptidomimetic macrocyle show improved long-term survivalcompared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe p53 MDM2 system, labeled stabilized peptidomimetic macrocyles basedon the p53 is used in an MDM2 binding assay along with small moleculesthat competitively bind to MDM2. Competitive binding studies allow forrapid in vitro evaluation and determination of drug candidates specificfor the p53/MDM2 system. Likewise in the BH3/BCL-X_(L) anti-apoptoticsystem labeled peptidomimetic macrocycles based on BH3 can be used in aBCL-X_(L) binding assay along with small molecules that competitivelybind to BCL-X_(L). Competitive binding studies allow for rapid in vitroevaluation and determination of drug candidates specific for theBH3/BCL-X_(L) system. The invention further provides for the generationof antibodies against the peptidomimetic macrocycles. In someembodiments, these antibodies specifically bind both the peptidomimeticmacrocycle and the p53 or BH3 peptidomimetic precursors upon which thepeptidomimetic macrocycles are derived. Such antibodies, for example,disrupt the p53/MDM2 or BH3/BCL-XL systems, respectively.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) BCL-2 family member expression or activity(e.g., extrinsic or intrinsic apoptotic pathway abnormalities). It isbelieved that some BCL-2 type disorders are caused, at least in part, byan abnormal level of one or more BCL-2 family members (e.g., over orunder expression), or by the presence of one or more BCL-2 familymembers exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of the BCL-2 family member or the enhancement ofthe level and/or activity of the BCL-2 family member, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing hyperproliferative disease by interfering with theinteraction or binding between p53 and MDM2 in tumor cells. Thesemethods comprise administering an effective amount of a compound of theinvention to a warm blooded animal, including a human, or to tumor cellscontaining wild type p53. In some embodiments, the administration of thecompounds of the present invention induce cell growth arrest orapoptosis. In other or further embodiments, the present invention isused to treat disease and/or tumor cells comprising elevated MDM2levels. Elevated levels of MDM2 as used herein refers to MDM2 levelsgreater than those found in cells containing more than the normal copynumber (2) of mdm2 or above about 10,000 molecules of MDM2 per cell asmeasured by ELISA and similar assays (Picksley et al. (1994), Oncogene9, 2523 2529).

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the peptidomimetics macrocycles of the invention isused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, myelodysplasia

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons, and the anti-apoptotic peptidomimetics macrocycles ofthe invention are used, in some embodiments, in the treatment of thesedisorders. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,spinal muscular atrophy, and various forms of cerebellar degeneration.The cell loss in these diseases does not induce an inflammatoryresponse, and apoptosis appears to be the mechanism of cell death. Inaddition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic peptidomimetics macrocycles ofthe invention are used to treat all such disorders associated withundesirable cell death.

Some examples of immunologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto organ transplant rejection, arthritis, lupus, IBD, Crohn's disease,asthma, multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto diabetes, hypothyroidism, hypopituitarism, hypoparathyroidism,hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, atherosclerosis, myocardialinfarction, stroke, thrombosis, aneurism, heart failure, ischemic heartdisease, angina pectoris, sudden cardiac death, hypertensive heartdisease; non-coronary vessel disease, such as arteriolosclerosis, smallvessel disease, nephropathy, hypertriglyceridemia, hypercholesterolemia,hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema andchronic pulmonary disease; or a cardiovascular condition associated withinterventional procedures (“procedural vascular trauma”), such asrestenosis following angioplasty, placement of a shunt, stent, syntheticor natural excision grafts, indwelling catheter, valve or otherimplantable devices. Preferred cardiovascular disorders includeatherosclerosis, myocardial infarction, aneurism, and stroke.

EXAMPLES

The following section provides illustrative examples of the presentinvention.

Example 1. Preparation of Alpha,Alpha-Disubstituted Amino Acids

Compound 1 was prepared by a two step sequence according to Yao et al.(2004) J. Org. Chem, 69:1720-1722. To 1-iodo-4-chloro-butane (1 ml, 8.2mmol) in dimethyl formamide (10 ml) was added sodium azide (0.53 g, 8.2mmol) and the reaction was stirred at ambient temperature overnight. Thereaction was then diluted with ethyl acetate and water. The organiclayer was washed with water (3 times), dried over magnesium sulfate andconcentrated in vacuo to give 1-azido-4-chloro-butane in 80% yield. Theazide was diluted with acetone (38 ml) and sodium iodide (1.98 g, 13.00mmol) was added and the reaction was heated at reflux overnight.Afterwards, the reaction was diluted with water and the product wasextracted with ether (three times). The combined organic extracts werewashed with sodium bicarbonate and brine. The organic extracts weredried over magnesium sulfate and concentrated in vacuo. The product 1was purified by passing it through a plug of neutral alumina. The yieldwas 89%.

Compound 2 was prepared by a three step sequence according to Belokon etat (1998), Tetrahedron Asymm. 9:4249-4252. A solution of S-proline (100g, 0.869 mol) and KOH (172 g, 2.61 mol) in isopropanol (600 ml) wasprepared with stirring at 40 C. As soon as the solution becametransparent, benzyl chloride (156 ml, 1.34 mol) was added at the sametemperature. After the addition was complete (3.5 h), the reaction wasstirred overnight at 40 C. The reaction was neutralized with conc. HCl(110 ml) until pH 5, then chloroform (400 ml) was added to the reactionmixture and the mixture was left stirring overnight. The mixture wasthen filtered and the precipitate washed with CHCl. The CHCL3 solutionswere combined and evaporated, the residue was treated with acetone andthe precipitate of the crude product filtered and additionally washedwith acetone. The benzyl proline product was isolated in 75% yield.

To a solution of benzyl proline (41 g, 0.2 mol) in methylene chloride(200 ml) was added thionyl chloride (18.34 ml, 0.25 mol) with stirringat −20 C to −30 C over a period of 10 min. The stirring was continued at−10 C until the reaction mixture became almost transparent. Then asolution of 2-aminobenzophenone (25 g, 0.125 mol) in methylene chloride(100 ml) was added to the reaction mixture at −30 C with stirring. Thestirring was continued at ambient temperature for another 10 h and asolution of sodium carbonate (40 g) in water (150 ml) was added to thereaction mixture with stirring at 0 C. The organic layer was separated,the aqueous layer extracted several times with methylene chloride andthe organic solutions were combined and evaporated. The product (benzylproline-aminobenzophenone adduct) was crystallized from ethanol and wasisolated in 85% yield.

A solution of KOH (23.1 g, 0.35 mol) in methanol (75 ml) was poured intoa stirred mixture of benzyl proline-aminobenzophenone adduct (19.5 g,0.05 mol) and nickel nitrate hexahydrate (29.1 g, 0.1 mol), L-Ala (8.9g, 0.1 mol) in methanol (175) under nitrogen at 40-50 C. The reactionmixture was stirred at 55 C-65 C for 2 h and then neutralized withacetic acid (20 ml). The reaction volume was diluted with water (500ml). After 6 h, the separated crystalline solid was filtered and washedwith water (2×) to give the pure compound 2 (red solid, 22 g). M+H obs.512.4, M+H calc. 512.1.

To compound 2 (5.122 g, 10.0 mmol) was added dimethyl formamide (45 mL),which was degassed via a freeze-thaw cycle. The solution was cooled to 4C with an ice bath and powdered KOH (6.361 g, 100 mmol) was added in onebatch. The cold bath was removed and compound 1 (3.375 g, 15 mmol)dissolved in dimethylformamide (4.0 mL) was added via syringe. Thereaction was stirred at ambient temperature for 40 min. The reaction wasthen quenched by adding it slowly to a cold solution of 5% aqueousacetic acid (200 mL). The crude product was collected by filtration andwashed three times with cold water. The product was purified by flashchromatography using a Biotage silica column and hexane ethyl acetate aseluent. Compound 3 was obtained as a red solid, (51% yield), M+H calc.609.2, M+H obs. 609.37. The purity was determined as 98% by UV 254 nm.

To a solution of 5-chloro pentyne (5 g, 47.8 mmol) in acetone (80 mL)was added sodium iodide (14.321 g, 95.54 mmol). The reaction was heatedat reflux overnight. Afterwards, the reaction was diluted with water andthe product was extracted with ether (three times). The combined organicextracts were washed with sodium bicarbonate and brine. The organicextracts were dried over magnesium sulfate and concentrated in vacuo.The product 4 was purified by passing it through a plug of neutralalumina. The yield was 92%.

To compound 2 (2.561 g, 5.0 mmol) was added dimethyl formamide (23 mL),which was degassed via a freeze-thaw cycle. The solution was cooled to 4C with an ice bath and powdered KOH (3.181 g, 50 mmol) was added in onebatch. The cold bath was removed and compound 4 (1.94 g, 10 mmol))dissolved in dimethylformamide (2.0 mL) was added via syringe. Thereaction was stirred at ambient temperature for 40 min. The reaction wasthen quenched by adding it slowly to a cold solution of 5% aqueousacetic acid (100 mL). The crude product was collected by filtration andwashed three times with cold water. The product was purified by flashchromatography using biotage silica column and hexane ethyl acetate aseluent. Compound 5 is a red solid, 1.4 g yield 48%. M+H calc. 578.19,M+H obs. 578.69. The purity was determined as 97% by UV 254 nm.

To a solution (12 ml) of 1/1 3N HCl/MeOH at 70 C was added a solution ofcompound 3 (1 g, 1.65 mmol) in MeOH (3 ml) dropwise. The startingmaterial disappeared within 5-10 min. The reaction mixture was thenconcentrated in vacuo and residual solvent removed on a high vacuumpump. The crude residue was diluted with 10% aqueous Na2CO3 (16 ml)cooled to 0 C with an ice bath. Fmoc-OSu (0.84 g, 2.5 mmol) dissolved indioxane (16 ml) was added and the reaction was allowed to warm up toambient temperature with stirring overnight. Afterwards, the reactionwas diluted with ethyl acetate and 1 N HCl. The organic layer was washedwith 1 N HCl (3 times). The organic layer was then dried over magnesiumsulfate and concentrated in vacuo. The pure product was isolated after aflash chromatography purification with a Biotage silica column and ethylacetate/hexane and methylene chloride/methanol as eluents to give aviscous oil in 36% overall yield for both steps. M+Na obs 431.89, M+Hcalc (409.18). Purity was determined as 98% UV 254 nm.

To a solution (18 ML) of 1/1 3N HCl/MeOH at 70 C was added a solution ofcompound 5 (1.4 G, 2.4 mmol) in MeOH (4 ml) dropwise. The startingmaterial disappeared within 5-10 min. The reaction mixture was thenconcentrated in vacuo and residual solvent removed on a high vacuumpump. The crude residue was diluted with 10% aqueous Na2CO3 (24 ml)cooled to 0 C with an ice bath. Fmoc-OSu (0.98 g, 2.9 mmol) dissolved indioxane (24 ml) was added and the reaction was allowed to warm up toambient temperature with stirring overnight. Afterwards, the reactionwas diluted with ethyl acetate and 1 N HCl. The organic layer was washedwith 1 N HCl (3 times). The organic layer was then dried over magnesiumsulfate and concentrated in vacuo. The pure product was isolated (30%yield for both steps) after flash chromatography purification with aBiotage silica column and ethyl acetate/hexane and methylenechloride/methanol as eluents. The product was obtained as a viscous oil,that solidifies upon standing. M+H calc. 378.16, M+Na obs 400.85.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A compound comprising: (a) a peptidomimeticmacrocycle; (b) an alkyne moiety; and (c) an amino acid sequencecomprising an amino acid moiety of the formula:

wherein: R₂ is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl, each of which is unsubstituted orsubstituted with halo-, or —H; each Q and T is independently selectedfrom the group consisting of alkylene, cycloalkylene,heterocycloalkylene, arylene, heteroarylene and [—R₄—K—R₄—]_(n), each ofwhich is unsubstituted or substituted with R₅; each K is independentlyO, S, SO, SO₂, CO, CO₂, or CONR₃; each R₃ is independently alkyl,alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which isunsubstituted or substituted with R₅, or —H; each R₄ is independentlyalkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,heterocycloalkylene, arylene, or heteroarylene; each R₅ is independentlyhalogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, afluorescent moiety, a radioisotope or a therapeutic agent; each R₆ isindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeuticagent; R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroalkyl, or heterocycloalkyl; each R₁₀ and R₁₁ is independently —Hor any protecting group suitable for peptide synthesis; each g and h isindependently an integer from 0 to 5; and n is an integer from 1 to 5,wherein the compound comprises an α-helix.
 2. The compound of claim 1,wherein R₂ is hydrogen.
 3. The compound of claim 1, wherein R₂ ismethyl.
 4. The compound of claim 1, wherein a secondary structure of thecompound is more stable than a corresponding secondary structure of acorresponding non-macrocyclic polypeptide.
 5. The compound of claim 1,wherein the α-helix comprises from 1 turn to 5 turns.
 6. The compound ofclaim 1, wherein the α-helix is more stable than an α-helix of acorresponding non-macrocyclic polypeptide.
 7. The compound of claim 1,wherein the α-helix comprises about 2 turns.
 8. The compound of claim 1,wherein the compound has an α-helical secondary structure, and thecompound is more stable than a corresponding α-helical secondarystructure of a corresponding non-macrocyclic polypeptide.
 9. Thecompound of claim 1, wherein the compound comprises a macrocyclic ringof about 29 atoms to about 37 atoms.
 10. The compound of claim 1,wherein the compound comprises an α-helix in aqueous solution.
 11. Thecompound of claim 10, wherein the compound exhibits increased α-helicalstructure in aqueous solution compared to a correspondingnon-macrocyclic polypeptide.
 12. The compound of claim 1, wherein thecompound exhibits increased thermal stability compared to acorresponding non-macrocyclic polypeptide.
 13. The compound of claim 1,wherein the compound exhibits increased biological activity compared toa corresponding non-macrocyclic polypeptide.
 14. The compound of claim1, wherein the compound exhibits increased resistance to proteolyticdegradation compared to a corresponding non-macrocyclic polypeptide. 15.The compound of claim 1, wherein the compound exhibits increased abilityto penetrate living cells compared to a corresponding non-macrocyclicpolypeptide.
 16. The compound of claim 1, wherein the compound comprisesa crosslink that spans from 1 turn to 5 turns of the α-helix.
 17. Thecompound of claim 16, wherein the crosslink spans 1, 2, 3, 4, or 5 turnsof the α-helix.
 18. The compound of claim 1, wherein the compoundcomprises a crosslink, wherein the crosslink is about 5 Å to about 9 Åin length per turn of the α-helix.